The Wnt/β-catenin pathway regulates diverse biological processes during development and tissue homeostasis through modulating the protein stability of β-catenin (Clevers et al., (2006) Cell 127:469-480; and Logan et al., (2004) Annu. Rev Cell Dev. Bial 20:781-810). In the absence of Wnt signaling, cytoplasmic β-catenin is associated with the β-catenin destruction complex that contains multiple proteins including adenomatous polyposis coli (APC), Axin, and glycogen synthase kinase 3 (GSK3). In this complex, β-catenin is constitutively phosphorylated by GSK3 and degraded by the proteasome pathway. The Wnt signal is transduced across the plasma membrane through two distinct receptors, the serpentine receptor Frizzled, and the single-transmembrane protein LRP5 or LRP6. The Wnt proteins promote the assembly of the Frizzled-LRP5/6 signaling complex, and induce phosphorylation of the cytoplasmic PPPSPxS motifs of LRP5/6 by GSK3 and Casein Kinase I. Phosphorylated LRP5/6 bind to Axin and inactivate the β-catenin degradation complex. Stabilized β-catenin enters the nucleus, binds to the TCF family transcription factors, and turns on transcription.
The large extracellular domain of LRP5/6 contains four YWTD-type β-propeller regions that are each followed by an EGF-like domain, and the LDLR domain. Each propeller region contains six YWTD motifs that form a six-bladed β-propeller structure. Biochemical studies suggest that Wnt proteins physically interact with both Frizzled and LRP6 and induce formation of Frizzled-LRP6 signaling complex (Semenov et al., (2001) Curr. Biol 11, 951-961; and Tamai, et al. (2000) Nature 407, 530-535). Besides Wnt proteins, the large extracellular domain of LRP5/6 binds to multiple secreted Wnt modulators, including Wnt antagonist, DKK1 and Sclerostin (SOST), and Wnt agonist R-Spondins.
Mutations in pathway components such as APC and β-catenin have been associated with human cancers. Recent studies suggest that overexpression of Wnt proteins and/or silencing of Wnt antagonists such as DKK1, WISP and sFRPs promote cancer development and progression (Akiri et al., (2009) Oncogene 28:2163-2172; Bafico et al., (2004) Cancer Cell 6:497-506; Suzuki et al., (2004) Nat Genet. 36:417-422; Taniguchi et al., (2005) Oncogene. 24:7946-7952; Veeck et al., (2006) Oncogene. 25:3479-3488; Zeng et al., (2007) Hum. Pathol. 38:120-133). In addition, Wnt signaling has been implicated for the maintenance of cancer stem cells (Jamieson et al., (2004) Cancer Cell 6:531-533 and Zhao et al., (2007) Cancer Cell 12:528-541).
Antibody therapy has been used as a means to treat certain cancers. Efforts to increase the valency or the number of antigenic determinants that an individual antibody molecule can bind have lead to the development of bispecific antibodies (for examples see Jimenez et al., Molecular Cancer Therapeutics 2005:4427-434, Lu et al., J. of Immun. Methods 1999:230, 159-171 and U.S. Patent Publication Nos. 20070014794 and 20050100543). Bispecific antibodies are immunoglobulin (Ig)-based molecules that bind to two different epitopes on either the same or distinct antigens. The antibodies, for example, could be specific for a tumor cell antigen and an effector cell such as an activated T-cell or two functional targets or epitopes.
A major obstacle in the development of bispecific antibodies as therapeutics has been difficulty in producing the antibodies in sufficient quantity and quality for clinical studies. In particular, traditional methods, including hybrid hybridoma, in which two distinct hybridomas are fused to create a cell expressing two sets of heavy and light chains, and chemical conjugation (Carter et al., (1995) J. Hematotherapy 4:463-70) have been inadequate. For example, coexpression of two different sets of IgG light and heavy chains in a hybrid hybridoma may produce up to 10 light- and heavy-chain pairs, with only one of these pairs forming the functional bispecific heterodimer (Suresh et al. (1986) Methods Enzymol. 121:210-28). In addition, purification of the antibodies from the non-functional species, such as homodimers and mispaired heterodimers of non-cognate Ig light and heavy chains produced by the hybrid hybridoma is cumbersome and inefficient.
Chemical crosslinking of two IgGs or their fragments is also inefficient and can lead to the loss of antibody activity (Zhu et al. (1994) Cancer Lett. 86:127-34). Multimeric aggregates resulting from chemical conjugation result in a poor yield (Cao et al. (1998) Bioconj. Chem. 9:635-44).
Accordingly, a need exists for functional multivalent antibodies capable of binding at least two or more epitopes with high affinity. In particular, there is need for functional multivalent antibodies that modify receptors with more then one modifying ligand, such as the canonical Wnt signaling co-receptor, LRP6.